Proteomics seeks to generate an identity profile of the entire proteome of an organism and, through analysis of this information, to identify potential diagnostic and therapeutic entities. Current technologies for resolving protein mixtures include two-dimensional gel electrophoresis and multi-dimensional liquid chromatography. Both of these techniques may be coupled to mass spectrometry. An example of this approach is the resolution and identification of 1,484 proteins in yeast (Washburn et al., Nat. Biotechnol. 19(3): 242-2471 (2001)). Another example of methodology that separates and identifies proteins is a modified version of the yeast two-hybrid screening assay developed by Uetz et al. (Uetz et al., Nature 403(6770): 623-627 (2000)) and Ito et al. (Ito et al., Proc. Natl. Acad. Sci. USA 98(8): 4569-4574 (2001)), which identified over 4,000 protein-protein interactions in Saccharomyces cerevisiae. A quantitative methodology for protein separation and identification is isotope coded affinity tag (ICAT), developed by Aebersold and colleagues (Smolka et al., Anal. Biochem. 297(1): 25-312 (2001)). ICAT involves site-specific, covalent labeling of proteins with isotopically normal or heavy reagents to quantitate levels of protein expression.
Complex protein mixtures may also be separated on libraries of combinatorially-generated ligands. Following exposure of an entity molecule to a combinatorial library, the entity may bind to ligands in the library. Detection of the bound entity may be accomplished when a purified, radiolabeled initial entity is used (Mondorf et al., J. Peptide Research 52: 526-536 (1998)). Other methods include detection by an antibody against the entity (Buettner et al., International Journal of Peptide & Protein Research 47: 70-83 (1996); Furka et al., International Journal Peptide Protein Research 37(6): 487-493 (1991); and Lam et al., (1991) supra). Ligands to multiple entities can be detected using beads immobilized on an adhesive in combination with a subtractive screening method. This is referred to as the QuASAR method (International (PCT) Patent Application WO 01/40265) and was used to detect ligands that bound to virus and prion protein.
FIoNA assay technology (Hammond et al International (PCT) Patent Application WO 04/007757) and other combinatorial techniques can identify a ligand:entity interation. The FIoNA assay technology identifies proteins from mixtures based on chemical, physical, biological, and/or biochemical function and not merely on their ability to bind a ligand within the library. Thus, the goal of FIoNA is to identify a ligand-support that binds a desired property, then to decode the ligand on the appropriate bead, and synthesize the bead in appropriate amounts to purify the one, or few proteins with the desired activity using current proteomic methods.
The full analysis of analytes in complex biological extracts is hindered by the large difference in concentration between individual analytes. In most biological mixtures some analytes are present at high concentration and others only present at trace-levels. As a result, the concentration of analytes may not be adapted to the dynamic range of a given analytical method. That is to say, the difference in the signal strengths produced by the most abundant and least abundant analyte species in a sample generally is wider than the ability of the analytical method to detect and accurately measure. For example, highly concentrated proteins may saturate the detection system and very low concentrations may be below the sensitivity of the analytical method, as occurs in human serum where the difference in concentration between the most abundant protein (albumin—tens of mg/ml) and the least abundant (e.g., IL-6—less than 1 pg/ml) may reach factors as high as hundreds of millions.
Two ways are currently followed to deal with this gap: the first is to design more adapted instruments and the second is to alter the sample for analysis.
One method of altering the sample is to deplete the sample of the more abundant species, thereby making the less abundant species more available for detection. This method involves, for example, the use of linker moieties, such as antibodies or specific dyes, that are directed to particular species in the sample. For example, in the case of plasma, the abundant proteins include albumin, immunoglobulins, fibrinogen, and alpha-1 proteinase inhibitor. Immunoaffinity columns are expensive, seldom totally specific for their target and will remove proteins associated with the target proteins. Moreover, once the most abundant proteins are removed, another set becomes the most abundant, which then creates the need to develop additional affinity columns. In addition, biological samples from different tissues within the same species and tissues from different species may have a completely different set of most abundant proteins. This method also suffers from the fact that elimination of some analyte species also eliminates species that interact with them. Thus some species that may be of interest are eliminated. While eliminating proteins of high abundance may help in some instances, this approach does not result in the detection of very low abundance proteins whose concentration is still below the sensitivity of the instrument to detect. Moreover, highly abundant species are represented by several proteins (even several dozen in some situations) and therefore a number of specific methods would have to be designed to address each different abundant species. Therefore, this method does not substantially compress the range of concentrations between the remaining analyte species.
Another method is to fractionate the sample, typically by chromatography. This method results in the compartmentalization of classes of analytes into different fractions based on similar biochemical properties. For example, ion exchange chromatography will compartmentalize proteins into fractions based on charge, while size exclusion chromatography compartmentalizes proteins based on size. Therefore, these methods may reduce the concentration range of the analytes, but at the cost of substantially decreasing the diversity of the population of analyte species within each compartment.